1. Field of the Invention
The present invention relates generally to a method of human gene therapy using an adeno-associated viral (AAV) vector. More specifically, the present invention relates to the use of an AAV More specifically, the present invention relates to the use of an AAV vector having an essentially wildtype phenotype as a human gene therapy vector for expressing a foreign, therapeutic gene of interest having a size of up to 1000 nucleotide base pairs. Additionally, the engineered essentially wildtype AAV vectors may be used as complementors in a method for generating defective AAV vectors.
2. Description of the Related Art
Adeno-associated virus is a helper-dependent human parvovirus which is able to infect cells latently by chromosomal integration. Various studies from 1970 to 1986 demonstrated that 15-30% of immortalized cells could be infected latently with wildtype AAV, and that the AAV genome was chromosomally linked (Hoggan et al., Proc. 4th Lepetite Colloquium, Cocoyac, Mexico, North-Holland, Amsterdam, pp 243-49, (1972).; Cheung et al., J. Virol. 33:739-48 (1980); Laughlin et al., J. Virol. 60:515-21 (1986)). Moreover, a similar ability for integration was demonstrated for recombinant AAV in immortalized tissue culture cells (Hermonat and Muzyczka, Proc. Natl. Acad. Sci. 81:6466-70 (1984); and Tratschin et al., Mol. Cell. Biol. 5:3251-60 (1985)). In 1988, recombinant AAV transduction of primary hematopoietic stem cells was achieved (LaFace et al., Virology 60:483-86 (1988)).
More recently, the preferred site of wildtype AAV integration was demonstrated to be in a region of human chromosome 19 (see, e.g., Kotin et al., Proc. Natl. Acad. Sci. U.S.A. 87:2211-15 (1991); Kotin et al., EMBO. J. 11:5071-78 (1990); and Samulski et al., EMBO J. 10:3941-50 (1991)). Many laboratories have confirmed and extended these data and demonstrated the utility of AAV-based vectors (see, e.g., McLaughlin et al., J. Virol. 62:1963-73 (1988); Lebkowski et al., Mol. Cell. Biol. 8:3988-96 (1988); Samulski et al., J. Virol. 63:3822-28 (1988); Zhou et al., Exper. Hematol. 21:928-33 (1993); Flotte et al., Gene Therapy 2:29-37 (1995); Russel et al., Proc. Natl. Acad. Sci. U.S.A. 92:5719-23 (1995); and Chiorini et al., Hum. Gene Ther. 6:1531-1541 (1995)).
In the last year, the mechanism of wildtype AAV integration was shown to involve a complex between chromosome 19 DNA and AAV terminal repeat DNA in the presence of the AAV Rep78/68 protein (Weitzman et al., Proc. Natl. Acad. Sci. U.S.A. 91:5808-12 (1994); Giraud et al., Proc. Natl. Acad. Sci. U.S.A. 91:10039-43 (1994); and Urcelay et al., J. Virol. 69:2038-46 (1995)). The same protein is required for AAV DNA replication (Hermonat et al., J. Virol. 51:329-39 (1984); and Tratschin et al.,. Mol. Cell. Biol. 5:3251-60 (1985)).
Because of its ability to integrate chromosomally and its nonpathogenic nature, adeno-associated virus (AAV) has significant potential as a human gene therapy vector. However, AAV has a disadvantage as a gene therapy vector due to its limited packaging capacity. Additional disadvantages of recombinant AAV vectors as gene therapy vehicles include the difficulty in producing high virus titers and the vector's inability, after essential genes are deleted, to integrate into chromosome 19 due to the lack of the rep gene (Rep78/68).
The prior art is deficient in that AAV vectors used for gene therapy have not had the ability to integrate into chromosome 19 or to generate high titers of virus. The present invention fulfills this long-standing need and desire in the art.